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Endocrinology Vol. 145, No. 6 2858-2864
Copyright © 2004 by The Endocrine Society

Effect of Ageing on Hormone Secretion and Follicular Dynamics in Sheep with and without the Booroola Gene

Antonio Gonzalez-Bulnes, Carlos J. H. Souza, Bruce K. Campbell and David T. Baird

Departamento de Reproduccion Animal, Instituto Nacional de Investigaciones Agrarias (INIA) (A.G.-B.), 28040 Madrid, Spain; Department of Obstetrics and Gynaecology, Centre for Reproductive Biology, University of Edinburgh Chancellor’s Building (A.G.-B., C.J.H.S., D.T.B.), Edinburgh, United Kingdom EH16 4SB; and the School of Human Development (B.K.C.), University of Nottingham, Queen Medical Centre, Nottingham NG7 2UH, United Kingdom

Address all correspondence and requests for reprints to: Antonio Gonzalez-Bulnes, Departamento de Reproduccion Animal, Instituto Nacional de Investigaciones Agrarias (INIA), Avenida Puerta de Hierro s/n, 28040 Madrid, Spain. E-mail: bulnes{at}inia.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been suggested that ewes carrying the Booroola gene (FecB) consistently ovulate more follicles because they recruit more primordial follicles and/or have a lower rate of atresia. If the former is correct, the pool of follicles would be depleted sooner in FecB animals. We have studied follicular dynamics and endocrine function during follicular and early luteal phases of the estrous cycle of older ewes with or without the fecundity gene and compared this data with data obtained 6 yr previously in the same animals. Older sheep carrying the Booroola gene maintained a significantly higher ovulation rate than noncarrier ewes [4.2 ± 0.8 vs. 2.2 ± 0.6 corpora lutea (CL), respectively; P < 0.05], and in keeping with data from young animals, both ovulatory follicles and CL (4.7 ± 0.3 vs. 6.9 ± 0.7 mm and 12.8 ± 0.5 vs. 16.7 ± 0.8 mm, respectively) were smaller than those of noncarrier ewes (P < 0.05). The interval from luteolysis to the onset of the LH surge increased with age in all the animals (from 52.0 ± 8.0 to 67.0 ± 7.5 h in gene carrier sheep and from 56.0 ± 2.0 to 79.5 ± 9.6 h in noncarrier sheep, P < 0.05). The concentration of estradiol and inhibin A in the early luteal phase was lower in older noncarrier ewes (P = 0.08 and P < 0.05, respectively), and the level of inhibin A was inversely related to the level of FSH in aged sheep of both genotypes (P < 0.0001). In contrast, the number of developing follicles in older ewes of both genotypes was similar to the number found in younger ewes, suggesting that increased ovulation rate in sheep carrying the FecB mutation is related to a reduced rate of atresia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EWES CARRYING THE Booroola autosomal mutation (FecB) have an increased ovulation rate and litter size (1). It was originally reported that these ewes had more follicles maturing and ovulating at a smaller diameter (2, 3) and higher concentrations of FSH in blood (4, 5). Souza et al. (6) used ewes with ovarian autotrasplant, a model with the advantage of allowing repeated collection of ovarian venous blood to determine secretory status of follicles observed by ultrasound. Results from this study confirm the higher number and the smaller diameter of preovulatory follicles in ewes with FecB. However, there were no differences in FSH levels or in the secretion of ovarian hormones between sheep carrying or not carrying the fecundity gene. This finding, together with the maintenance of differences in number and size of preovulatory follicles in ewes made hypogonadotropic by GnRH antagonist treatment and stimulated with exogenous gonadotropins, suggested that the Booroola gene exerts control of follicle growth at the level of the ovary (7).

The most consistent characteristic of ewes carrying the FecB gene is the precocious development of a higher number of ovulatory follicles associated with an earlier proliferation and differentiation of granulosa cells (8, 9, 10). Differences in the number of preovulatory follicles and ovulation rate are associated with a higher number of large growing follicles both during the follicular and the luteal phase of the cycle (6). The explanation of the mechanisms involved for such differences in the number of preovulatory follicles and ovulation rate between prolific and nonprolific sheep breeds/genotypes has been the target of many studies in the last 30 yr (11). Reduced follicular diameter associated with a higher number of ovulatory follicles has been described in many prolific breeds such as Finnish Landrace (12) and Romanov (13). The following two main reasons were proposed for increased ovulation rate: an increased rate of recruitment of follicles into the growing pool or a lower rate of follicular atresia (14).

The objective of current study was to elucidate the mechanisms for the increased follicular population and ovulation rate in Booroola gene carriers by determining the effect of age and genotype on the ovarian-pituitary axis. We had available a group of experimental ewes with ovarian autotransplant that had been first studied in 1996 when they were 4–6 yr old (6). Six years later, in 2002, we measured the dynamics of follicular development by serial ultrasound and the concentrations of FSH, LH, estradiol, and inhibin A in ovarian venous blood during stages of the estrous cycle equivalent to the prior study. Assuming that the pool of primordial follicles at puberty is similar in gene carrier and noncarrier ewes (15), differences in the number of developing follicles in older ewes may indicate differences in follicular recruitment throughout their lifetime.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Ten Scottish Blackface x Merino ewes (8–14 yr old) with the left ovary autotransplanted to the neck were studied; four carried the FecB gene (two homozygous ewes, FecB FecB; and two heterozygous ewes, FecB Fec+), and six were noncarriers. The genotype of the sheep was determined in the prior study done in 1996 and again in 2002. First, the genotypes were determined by analyzing records of the ovulation rate and pedigree (1). Subsequently, the genotypes were confirmed by sequencing the bone morphogenetic receptor type 1 B gene (16) or by forced restriction fragment length polymorphism analysis (17). These ewes were hemiovariectomized; the right ovary was removed at the time of the original autotransplantation when they were 2–4 yr old. Sheep were housed indoors at the Marshall Building facilities (Roslin, Midlothian, Edinburgh, UK) and received a maintenance diet of hay and pelleted ration with water ad libitum. The experiments were performed under Project License PPL 60/1523, approved by the Home Office United Kingdom under the Animals (Scientific Procedures) Act 1986.

The study, conducted during the breeding season (October 2002), covered the follicular and the subsequent early luteal phase of the estrous cycle. Because ewes with autotransplanted ovaries do not cycle spontaneously due to the lack of direct communication between uterus and ovary (18), initiation and synchronization of the estrous cycle were achieved with two injections of cloprostenol, a potent analog of prostaglandin F2{alpha} (125 mg, im, Estrumate; Cooper’s Animal Health, Crewe, UK), given 13 d apart. The estrous cycle evaluated in the experiment was induced by a third injection of cloprostenol 14 d later. On the day before the injection of the third cloprostenol dose, ovarian veins were cannulated with silastic catheters (0.8 mm x 1.7 mm, internal and external diameters, respectively) under local anesthesia (2 ml sc of Lignocaine 2%; Lignavet, Leyland, UK) as previously described (19). The ewes were placed in metabolism crates and received im prophylactic antibiotic treatment (Duphapen; Solvay Duphar, Southampton, UK), repeated every 3 d throughout the experiment.

The dynamics of follicle development and endocrine changes around ovulation were studied in these same 10 ewes in 1996 using identical methods to those used in the present study (6). To assess the effect of age, these results were compared with the results in the same animals that were now 6 yr older.

Ovarian ultrasound scanning
The skin over the transplanted ovary was clipped and shaved at the beginning of the experiment and maintained free of wool throughout. Before each scan, the area was covered with scanning gel (Siel Sound Gel; Siel Imaging Equipment, Aldermaston, UK). The ovaries were scanned, using a 7.5-MHz linear transducer (model UST-5512U-7.5; Aloka, Tokyo, Japan) with a real-time ultrasound scanner (SSD-500, Aloka), on the day before the injection of the third cloprostenol dose and then at 12-h intervals from d 0–9 of the cycle. Every scan examined both horizontal (dorso/ventral) and vertical (cranio/caudal) planes, and images were recorded on videocassette tape for subsequent analysis. The tapes were played in slow motion, and the image was frozen at the largest section of the antral cavity for each individual follicle greater than 2.5 mm in diameter. The image was digitalized into pixels of 256 shades of gray, and using the NIH Image software (http://rsb.info.nih.gov/NIH-Image/index.html; National Institutes of Health, Bethesda, MD), the periphery of the follicle was identified. Measurements were taken for the major and minor axes of the best-fitted ellipsis for each follicle. The diameters of the follicles were determined as a mean of the two axes measured.

Hormone assays
Samples of venous blood (10 ml) were collected at 6-h intervals for 72 h from induction of luteal regression with the third cloprostenol injection and every 12 h from d 3–9 after cloprostenol. After sampling, each cannula was flushed with 5 ml of a solution of 250 IU sodium heparin/ml isotonic saline. The blood was centrifuged at 4 C, and the plasma was separated and stored at –20 C until assayed for gonadotropin (FSH and LH), inhibin A, and estradiol.

Gonadotropins were measured in duplicate using previously described double-antibody RIA for FSH (19) and LH (20). The sensitivities of the assays were 0.3 ng/ml for FSH and 0.2 ng/ml for LH. The intra- and interassay coefficients of variation were 7.2 and 10.1% for FSH and 9.4 and 12.3% for LH, respectively. Inhibin A was analyzed by enzyme-linked immunoassay (21). Sensitivity was 15 pg/ml and intra- and interassay coefficients of variation were 7.6 and 11.9%, respectively. Estradiol was measured in plasma using a RIA kit (Serono Diagnostics Ltd, Woking, Surrey, UK), which was validated for use in ovine plasma (22) and modified to increase sensitivity (23). Sensitivity of the assay was 0.5 pg/ml and the intra- and interassay coefficients of variation were 8.3 and 9.8%, respectively.

Statistical analysis
The data were normalized with respect to two time periods of physiological significance. The first was the time of the third cloprostenol injection, and the second was the time of onset of the LH surge, which was defined as the nadir point before LH concentrations exceeded 10 ng/ml (d 0). For analysis of the relationship between the diameters of the large follicles (that grew to a diameter of at least 4.5 mm) that developed during the luteal phase and hormone secretion, follicles were aligned by emergence (first time a follicle was observed in the scans with a diameter between 2.5 and 3 mm). When more than one large follicle or corpus luteum (CL) per ewe was observed, data from all structures were included to calculate the mean values. The correlation coefficients between time, follicular population, and endocrine profiles were analyzed by the Pearson correlation procedure; correlations were considered to be statistically significant at P < 0.05. The differences on the effect of time on number of total follicles, diameter of large follicles, and hormone concentrations and the comparison of the interval luteolysis-onset LH surge and the ovulation rate between the genotypes were tested by ANOVA. Thereafter, to evaluate the effect of age and interaction with genotypes, a retrospective analysis using ANOVA compared data obtained in the current study with data collected in 1996, 6 yr previously, at equivalent stages of the cycle from the same ewes. Data were expressed as mean ± SEM, and differences were considered to be statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experimental animals
Two noncarrier ewes were excluded from subsequent analysis. The first sheep was presumed to be anestrus because neither the CL from the previous cycle nor LH surge or ovulation was detected in response to the administration of cloprostenol. The other ewe showed a luteinized unruptured follicle cyst (maximum diameter, 18.9 mm) that persisted during the entire period of observation. Thus, the results are available from four FecB carriers (ages 8–13 yr) and four nongene carriers (ages 8–12 yr).

Patterns of follicular development in older ewes
The anticipated differences in the dynamics of follicle development between ewes with and without the fecundity gene, which had been reported previously in these ewes, persisted as they became older.

The ovulation rate was significantly higher in gene carriers than in noncarrier animals (4.2 ± 0.8 vs. 2.2 ± 0.6 CL; P < 0.05). The mean diameters of the follicles, which eventually ovulated in gene carriers, were significantly smaller (P < 0.05) in gene carriers than those in noncarrier animals, reaching a mean diameter of 4.7 ± 0.3 and 6.9 ± 0.7 mm, respectively, at the onset of the LH surge (Fig. 1AGo) and 6.3 ± 0.8 and 7.1 ± 0.6 mm, respectively, at the time of estimated ovulation on d 1 (Fig. 1BGo). Ovulatory follicles in gene carrier ewes gave rise to CL of smaller diameter (P < 0.05). In the early luteal phase, on d 6, the size of the ovulatory follicle/CL reached a mean diameter of 16.7 ± 0.8 mm in noncarrier and 12.8 ± 0.5 mm in carrier sheep.



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  		FIG. 1. Mean (± SEM) concentrations of LH (A), growth of the preovulatory follicles/CL and large antral follicles from the first wave (B), number of follicles >= 2.4 mm (C), and concentrations of inhibin A (D), and estradiol (E) in ovarian venous blood, and FSH (F) in jugular samples, during the follicular and the early luteal phase in older ewes carrying the FecB gene (filled squares) or not carrying the gene (open squares). Data (n = 4 per point) have been grouped around the time of injection of cloprostenol (PG) and the time of beginning of the LH surge (dotted line). Note the break in the scale. The mean diameter of the ovulatory follicles and the CL are significantly smaller (P < 0.05) in the FecB ewes.

 
Coincidental with the growth of the ovulatory follicles in the follicular phase, between the time of injection of cloprostenol and the time of estimated ovulation, there were progressive decreases in the total number of follicles >= 2.5 mm (Fig. 1CGo), reaching the lowest value around 36–48 h after the LH peak, with decreases from the maximum of 3.5 ± 0.1 to 1.2 ± 0.7 mm (P < 0.05) for carriers and 3.0 ± 0.6 to 1.0 ± 0.4 mm (P < 0.0001) in noncarriers. Thereafter, the number of follicles increased linearly during the early luteal phase (P < 0.05), when all ewes developed at least one large follicle. These follicles grew to reach a diameter of about 5 mm on d 3 and, thereafter, started to decrease around d 5. During the growing phase, the mean diameter of large follicles was slightly higher in noncarrier compared with carrier ewes (5.2 ± 0.1 vs. 4.8 ± 0.1 mm, respectively).

Patterns of hormone secretion in older ewes
There were no major differences in the pattern of hormone changes observed between the genotypes. The mean secretion of dimeric inhibin A was not different between the genotypes and remained relatively stable during the follicular phase (Fig. 1DGo). Inhibin A concentration decreased sharply from the LH surge to 60 h after in both genotypes (P < 0.005) and then showed a slight increase to remain stable at around 280 pg/ml. In both genotypes, the concentrations of inhibin A in ovarian venous blood were correlated with the number of antral follicles >= 2.5 mm (P < 0.01) and with the growth of large follicles >= 4.5 mm (P < 0.05). There were no significant differences in the mean concentrations of estradiol between gene carrier and noncarrier ewes (Fig. 1EGo). The secretion of estradiol increased after cloprostenol injection and reached the highest value around the LH peak in both genotypes, and thereafter, between d 3 and 5.5, a smaller rise in estradiol levels was detected.

The concentration and pattern of FSH during the periovulatory period were similar between the genotypes, although the mean level was 2- to 3-fold higher than that reported in young intact ewes using the same assay (Fig. 1FGo). In all the sheep, the concentration of FSH decreased after cloprostenol-induced luteolysis (P < 0.005) until the time of the LH surge, when it sharply increased until d 5 after the LH peak (P < 0.001). Decreases in FSH levels were inversely related to the growth of large follicles >= 4.5 mm (P < 0.005) and to the level of inhibin A (P < 0.0001) but not estradiol. The secretions of inhibin A and estradiol during the period of study were correlated in both genotypes (P < 0.01 for carrier and P < 0.001 for noncarrier ewes).

Effect of age on ovarian and endocrine function
There was no effect of age on ovulation rate in either gene carrier sheep (4.0 ± 1.2 and 4.2 ± 0.8 CL for young and old ewes, respectively) or in noncarrier sheep (2.7 ± 0.3 and 2.2 ± 0.6 CL for young and old ewes, respectively). The mean sizes of ovulatory follicles/CL during the periovulatory period and the early luteal phase were similar between different ages and genotypes. The same was found for the large follicles from the first wave of development. However, there were several differences in endocrine and in the patterns of follicular development in the preovulatory period in older ewes compared with when they were younger.

Although ovulatory follicles were of similar size in old and young animals just before ovulation, they were smaller at the time of recruitment when the cloprostenol injection was given in older ewes of both genotypes (Fig. 2AGo). These differences reached statistical significance in ewes not carrying the FecB gene (4.5 ± 0.3 vs. 2.7 ± 0.1 mm for noncarriers, P < 0.05) but not in gene carriers (3.6 ± 0.3 vs. 2.6 ± 0.6 mm for carriers). The mean concentration of FSH was significantly elevated in older ewes of both genotypes at all stages of the cycle (Fig. 2BGo, P < 0.0005). However, the pattern remains similar to that in younger ewes, with a fall during the follicular phase (P < 0.05), an ovulatory peak (P < 0.005), and a rise during the early luteal phase.



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  		FIG. 2. Mean diameter of the preovulatory follicles/CL and large antral follicles from the first wave (A), concentrations of FSH (B) in jugular samples, and concentrations of inhibin A (C) and estradiol (D) in ovarian venous blood during the follicular and the early luteal phase in younger (circles) and older ewes (squares) carrying the FecB gene (filled symbols) or not carrying the gene (open symbols). Data (n = 4 per point) have been grouped around the time of injection of cloprostenol (PG) and the time of beginning of the LH surge (dotted line). Note the break in the scale. SEMs have been omitted for clarity of the figure. The concentration of FSH is significantly higher in older ewes of both genotype (P < 0.0005), and there is no significant difference between genotype in younger or older ewes. On the other hand, the inhibin A concentrations are significantly lower in older nongene carrier ewes (P < 0.05) in the early luteal phase (d 1–6).

 
The interval from cloprostenol-induced luteolysis to the onset of the LH surge increased with age in all the animals (P < 0.05), changing from 52.0 ± 8.0 to 67.0 ± 7.5 h in gene carrier ewes and from 56.0 ± 2.0 to 79.5 ± 9.6 h in noncarrier ewes. Onset of LH surge and ovulation of follicles were associated with a decrease in inhibin A ovarian levels in both genotypes and ages (Fig. 2CGo). Patterns of secretion of inhibin were very similar between young and old ewes carrying the FecB gene, with a striking decrease occurring after the ovulatory surge of LH. In young noncarrier ewes, the rise in inhibin concentration in the early luteal phase of the cycle was significantly greater than in older ewes (P < 0.05). On the other hand, there was no statistically significant difference in the levels of estradiol between young and old animals (Fig. 2DGo), although the mean estradiol concentration tended to be higher in noncarrier young ewes (P = 0.08).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results indicate that older sheep carrying the Booroola gene still maintain the most consistent characteristic associated with the FecB mutation (i.e., an increase in ovulation rate). The ovulatory follicles were smaller and gave rise to smaller CL than those of ewes not carrying the fecundity gene. The processes of folliculogenesis and ovulation in older ewes were affected by the Booroola genotype in the same way described in early studies reporting reproductive characteristics of the sheep carrying the gene (24, 25) and even in the same ewes 6 yr previously (6). The first studies (1) described that the FecB gene increases the number of ovulations to three or four in heterozygote FecB Fec+ ewes (carriers of one mutated allele) and to five or more in homozygote FecB FecB ewes (carriers of two mutated alleles) compared with one or two ovulations in Fec+ Fec+ ewes (noncarriers of the mutation). This increase in ovulation rate is associated with a decrease in the size of preovulatory follicles (26) and an earlier loss of proliferative activity of granulosa cells, a high sensitivity to FSH and LH, and an earlier secretion of estradiol (2, 27). In the current study, there were no differences in ovulation rate and mean sizes of ovulatory follicles/CL during the periovulatory period and the early luteal phase in older ewes despite an overall increase in the concentration of FSH. However, ovulatory follicles were smaller at the time of recruitment in older ewes, and together with the delay of the onset of LH surge, this probably reflects the fact that, due to the reduced pool of follicles in older ewes, the chances of having at least one large healthy follicle available for recruitment at the time of luteal regression are much reduced. This hypothesis is supported by the reduced secretion of inhibin observed in older ewes during the luteal phase and the increased level of FSH, which is consistent with a decrease of the number and function of follicles with age. This decrease could be enhanced by the fact that the ewes were hemiovariectomized during the ovarian transplant procedure, which reduced the follicular population by half. Similar changes in hormone patterns have been observed at an earlier age in ewes in which the pool of follicles has been drastically reduced after autotransplantation of frozen ovarian cortical strips (28, 29). The same effect was previously described in a study comparing follicular and endocrine events in young and older nonprolific hemiovariectomized Finn-Merino sheep (30). In that experiment, despite the depletion of follicles, the ovulation rate was not affected by ageing. This fact suggests that the raised levels of FSH allow the development of most of the follicles, with a higher proportion of follicles reaching the ovulation. These findings are confirmed by results found in the current study, which show that the increased levels of FSH with age are related to a decrease in inhibin A rather than a change in estradiol secretion. It is accepted that the secretion of FSH is controlled by the negative feedback effect of both inhibin and estradiol (31). However, it has been suggested that inhibin regulates the basal concentration of FSH while estradiol regulates fluctuations of FSH that occur during the cycle (32). In the present study, the low levels of FSH occurred during the follicular phase when there was a large number of follicles >= 2.5 mm in size secreting both estradiol and inhibin A (33, 34, 35).

Changes in endocrine secretion and follicular dynamics were related to ageing, but excepting those characteristics of the Booroola mutation, there were no differences between carrier and noncarrier ewes with the same ages. Although we could demonstrate no difference in signs of incipient ovarian failure between genotype at the age studied (8–14 yr), it is possible that ovarian failure may have occurred earlier in FecB animals if the ewes had been studied through to the complete cessation of reproductive activity (probably about 14–20 yr). The lack of differences in FSH patterns and concentrations, in agreement with other previous studies (6, 36, 37, 38), reinforces the hypothesis that FecB gene modulates folliculogenesis and ovulatory processes at the level of the ovary (7). The lack of differences in the number of developing follicles and the secretion of inhibin, estradiol, and FSH between older sheep carrying or not carrying the fecundity gene points to a similar number and functionality of follicles and indicates that increased ovulation rate in sheep carrying the FecB gene may be more related to a reduced rate of atresia rather than to an increased follicular recruitment during lifetime (8). This reduction in atresia rates in highly prolific sheep with or without the fecundity gene is in agreement with previous data comparing atresia of follicles from Booroola gene carriers and noncarriers during last days of cycles synchronized with two doses of prostaglandin F2{alpha} (39). These results also support data from other studies indicating that ovulation rate in monovular sheep is more likely to be limited by high levels of atresia rather than by a lower recruitment of follicles into the growing pool (40, 41). A possible explanation of resistance to atresia in follicles from Booroola gene carriers may be related to the earlier differentiation of granulosa cells and acquisition of LH receptors (9) because regression of follicles is determined by an inability to grow in response to LH when plasma FSH levels are decreased (42, 43). An alternative explanation would be related to the regulation of ovarian folliculogenesis by changes in bioavailability of IGF and expression of IGF binding proteins (IGFBPs). Increases in IGFBP reduce IGF availability, inducing follicular atresia (44), and intrafollicular IGFBP is decreased in Booroola gene carriers (45).

In conclusion, the difference in patterns of follicular development and in the number of ovulatory follicles observed in ewes carrying the Booroola mutation compared with nongene carriers is maintained in older ewes. The levels of FSH are increased in older ewes, and this, together with lower inhibin A levels, reflects follicular depletion. There were very few differences between genotypes in their response to age, indicating an effect of the FecB gene at the ovarian level more likely than at the pituitary level. The lack of differences in the number of developing follicles between older sheep carrying or not carrying the Booroola mutation indicates that the effect of the FecB gene at the ovary may be more related to a reduced rate of atresia rather than to an increased follicular recruitment during lifetime.


   Acknowledgments
 
We gratefully acknowledge Mrs. J. Docherty, Ms L. Harkness, and Mr. N. Hollow for skilled technical assistance.


   Footnotes
 
This work was supported by the Programa "Ramon y Cajal" in Spain and by a fellowship under the Organisation for Economic Co-operation and Development (OECD) Co-operative Research Programme "Biological Resource Management for Sustainable Agriculture Systems" in the United Kingdom. The research was supported by Programme Grant G9827407 from the Medical Research Council.

Present address for C.J.H.S.: EMBRAPA Genetic Resources and Biotechnology, CEP 70770-900 Brasilia, Brazil.

Abbreviations: CL, Corpus luteum; FecB, Booroola gene; IGFBP, IGF binding protein.

Received December 19, 2003.

Accepted for publication March 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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